1. Field of the Invention
The present invention concerns a cDNA clone (.lambda.RAL-1) which encodes a protein with an apparent molecular weight of 42,000 in vivo and which clone reacts strongly with O. volvulus antisera. The invention also relates to a recombinant antigen produced by this clone, which stimulates T cells from infected individuals to proliferate. Still further, the invention is directed to an O. volvulus peptide sequence which contains three repeats of polypeptide sequence KKPEDWD.
2. Background Information
Onchocerciasis, or river blindness, is a major cause of infectious blindness in the world. In severely affected areas, as many as half of the adult males may be blinded by the disease (World Health Organization, Edpidemology of Onchocerciasis, WHO Tech. Rep. Ser. 597.: 1-99, (1976)), and mortality rates in such persons are increased as much as four fold (B. Kirkwood, P. Smith, T. Marshall and A. Prost, "Relationships Between Mortality, Visual Acuity and Microfilarial Load in the Area of the Onchocerciasis Control Program", Trans. Roy. Soc. Trop. Med. Hyg., 77: 862-863, (1983)). Attempts to control the disease have centered on control of the vector for the parasite, the blackfly Simulium sp., as well as on the development of chemotherapeutic agents which may be used to eliminate the parasite from infected individuals. Unfortunately, control of the vector has proven extremely difficult. Furthermore, to date no practical chemotherapeutic agent has been identified which kills the adult form of the parasite.
Because neither vector control nor chemotherapy has proven successful in controlling the disease, the development of a vaccine against infection with the parasite has become a high priority (Strategic Plan for Onchocerciasis Research, Edna McConnell Clark Foundation, N.Y., N.Y., (1985)). In order to develop a vaccine, it is necessary to first identify antigens which induce immunity against the parasite. In the case of onchocerciasis, however, the identification of such antigens is complicated by the fact that much of the pathology seen during the course of the disease may be caused by the induction of the immune response of the host to certain antigens of the parasite (C. D. MacKenzie, J. F. Williams, B. M. Sisley, M. W. Steward and J. O'Day, "Variations in Host Responses and the Pathogenesis in Human Onchocerciasis", Rev. Info Dis., 7: 802-808, (1985)). Because of this concern, it is necessary to delineate as fully as possible which parasite antigens are involved in inducing potentially beneficial, as opposed to harmful aspects of the host immune response.
In order to undertake such a dissection of the immune response, large quantities of purified parasite antigens are required. However, no animal host has been identified which can be used to provide the various stages of O. volvulus in quantity. As a consequence, the lack of parasite material has posed a major impediment to immunological studies. This problem has been particularly acute for the study of the infectious form of the parasite, the third stage larva, or L3. This is due to the fact that the infective larvae must be obtained by dissection of infected blackflies, which are extremely difficult to obtain. Antigens from this third stage larva may be especially important, since it has been shown in other filarial infections that irradiated infective larvae can induce protective immunity (M. M. Wong, M. F. Guest and M. J. Laviopierre, "Dirofilaria immitis: Fate and Immunogenicity of Irradiated Infective Stage Larvae in Beagles", Exp. Parasitol., 35: 465-474, (1974); J. A. Yates, and G. I. Higashi, "Brugia malayi: Vaccination of Jirds with .sup.60 Cobalt-Attenuated Infective Stage Larvae Protects Against Homologous Challenge", Am. J. Trop. Med. Hyg., 34: 1132-1137 (1985)).
______________________________________ DEFINITIONS Genbank: NIH Genetic Sequence Data Bank BNRF: National Biomedical Research Foundation Protein Data Bank Amino Acid Abbreviations Three One Amino Acid Letter Abbreviation Letter Abbreviation aspartic acid Asp D asparagine Asn N threonine Thr T serine Ser S glutamic acid Glu E glutamine Gln Q proline Pro P glycine Gly G alanine Ala A cysteine Cys C valine Val V methionine Met M isoleucine Ile I leucine Leu L tyrosine Tyr Y phenylalanine Phe F lysine Lys K histidine His H arginine Arg R Nucleic Acid Abbreviations T thymidine A adenine G guanine C cytosine U uracil ______________________________________
Nucleotide - A monomeric unit of DNA or RNA containing a sugar moiety (pentose), a phosphate, and a nitrogenous heterocyclic base. The base is linked to the sugar moiety via the glycosidic carbon (1' carbon of the pentose) and that combination of base and sugar is called a nucleoside. The base characterizes the nucleotide. The four DNA bases are adenine ("A"), guanine ("G"), cytosine ("C"), and thymine ("T"). The four RNA bases are A, G, C and uracil ("U").
DNA Sequence - A linear array of nucleotides connected one to the other by phosphodiester bonds between 3' and 5' carbons of adjacent pentoses.
Codon - A DNA sequence of three nucleotides (a triplet) which encodes through mRNA an amino acid, a translation start signal or a translation termination signal. For example, the nucleotide triplets TTA, TTG, CTT, CTC, CTA and CTG encode for the amino acid leucine ("Leu"), TAG, TAA and TGA are translation stop signals and ATG is a translation start signal.
Reading Frame - The grouping of codons during translation of mRNA into amino acid sequences. During translation, the proper reading frame must be maintained. For example, the sequence GCTGGTTGTAAG may be translated in three reading frame or phases, each of which affords a different amino acid sequence
GCT GGT TGT AAG AAG - Ala-Gly-Cys-Lys PA1 G CTG GTT GTA AG - Leu-Val-Val PA1 GC TGG TTG TAA G - Trp-Leu-(STOP).
Polypeptide - A linear array of amino acids connected one to the other by peptide bonds between the alpha-amino and carboxy groups of adjacent amino acids.
Genome - The entire DNA of a cell or a virus. It includes inter alia the structural genes coding for the polypeptides of the cell or virus, as well as its operator, promotor and ribosome binding and interaction sequences, including sequences such as the Shine-Dalgarno sequences.
Structural Gene - A DNA sequence which encodes through its template or messenger RNA ("mRNA") a sequence of amino acids characteristic of a specific polypeptide.
Transcription - The process of producing mRNA from a structural gene.
Translation - The process of producing a polypeptide from mRNA.
Expression - The process undergone by a structural gene to produce a polypeptide. It is a combination of transcription and translation.
Plasmid - A non-chromosomal double-stranded DNA sequence comprising an intact "replicon" such that the plasmid is replicated in a host cell. When the plasmid is placed within a unicellular organism, the characteristics of that organism may be changed or transformed as a result of the DNA of the plasmid. For example, a plasmid carrying the gene for tetracycline resistance (Tet.sup.R) transforms a cell previously sensitive to tetracycline into one which is resistant to it. A cell transformed by a plasmid is called a "transformant".
Phage or Bacteriophage - Bacterial virus, many of which consist of DNA sequences encapsulated in a protein envelope or coat ("capsid protein").
Cloning Vehicle - A plasmid, phage DNA or other DNA sequence which is capable of replicating in a host cell, which is characterized by one or a small number of endonuclease recognition sites at which such DNA sequences may be cut in a determinable fashion without attendant loss of an essential biological function of the DNA, e.g., replication, production of coat proteins or loss of promoter or binding sites, and which contains a marker suitable for use in the identification of transformed cells, e.g., tetracycline resistance or ampicillin resistance. A cloning vehicle is often called a vector.
Cloning - The process of obtaining a population of organisms or DNA sequences derived from one such organism or sequence by asexual reproduction.
Recombinant DNA Molecule or Hybrid DNA - A molecular consisting of segments of DNA from different genomes which have been joined end-to-end outside of living cells and have a capacity to infect some host cell and be maintained therein.
Expression Control Sequence - A sequence of nucleotides that controls and regulates expression of structural genes when operatively linked to those genes. They include the lac system, the trp system, major operator and promoter regions of phage .lambda., the control region of fd coat protein and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells of their viruses.
Transformation/Transfection - DNA or RNA is introduced into cells to allow gene expression.
"Infected" refers to the introduction of RNA or DNA by a viral vector into the host.
"Injected" refers to the microinjection (use of a small syringe) of DNA into a cell.